The development of rapid, low cost, point-of-care (POC) approaches for the quantitative detection of multiple proteins or biomarkers such as antibodies would drastically impact global health by allowing more frequent testing and by improving the penetration of molecular diagnostics into the developing world. Up to date, current methods for the quantitative detection of proteins, such as antibodies, still mostly rely on ELISAs, Western Blots and polarization assays, which are multi-step, wash- and reagent-intensive processes that necessitate specialized technicians and require several hours before completion. Alternative point-of-care approaches, such as immunochemical dipsticks, display important advantages in terms of ease-of-use and affordability. Unfortunately, however, these remain hardly multiplexable and their results are mostly qualitative thus open to subjective interpretation.
Over the last decade, two main categories of biosensing devices have been explored in order to achieve multiplexed, quantitative, point-of-care (POC) protein detection: lab-on-chip devices and single-step homogeneous assays. Lab-on-a-chip devices (e.g. microfluidic devices), which integrate and automate multiple steps onto a unique device, have shown great promise for POC multiplex detection in recent years. However, their high cost, requirement for reagents and relatively complex handling still provides important hurdles for their transition into the real world. Single-step homogeneous assays, on the other hand, which necessitate simple mix and read procedure without the necessity to process samples by separation or washing steps, represent another promising avenue for point-of-care biosensing. Unfortunately, however, few homogeneous assays are selective enough to enable the single-step detection of protein markers directly in unprocessed whole blood.
Numerous single-step homogeneous assays for protein detection have been developed in recent years. Those methods include protein-based assays, and DNA-based assays. Among these different approaches, DNA-based electrochemical sensors (E-DNA) have shown great promise for the transition into the real world. These sensors are typically rapid, reagent-less, multiplexable and versatile, allowing detection of nucleic acids, proteins and small analytes. They take advantage of the high specificity and programmability of DNA in addition to permitting easy electrochemical measurements directly in complex samples such as blood serum using relatively inexpensive potentiostats. Unfortunately, however, these sensors still display limitations that preclude their transition into real life applications. These include rapid signal drifts in the first minutes when the sensor is immersed in whole blood and low nano-amp electrochemical signal output.
A versatile DNA-based electrochemical switch was further developed that supports the rapid, quantitative detection of large macromolecules such as antibodies directly in whole blood at clinically relevant, low-nanomolar concentrations (Vallée-Bélisle et al., 2012). Unfortunately, however, this DNA switch only performs well at low surface density on the sensor head, a condition required in order to ensure that each single switch is activated by a large macromolecule that often span up to dozens of nm (e.g., antibodies: 12 nm). These low density sensors therefore only generate low nA/cm2 current densities, which can only be detected by expensive, laboratory-grade potentiostats. Furthermore, these switch-based DNA sensors remain relatively hard to synthetize since they require the addition of many chemical moieties (e.g. electro-active elements, C6-thiol, frame-inversion, epitopes . . . ) and typically require long equilibration time (minutes) in their working media (for example, whole blood) before they are stable enough to enable precise measurements.
Various technologies for detecting various targets were developed but their applications are limited. International application WO 2012/071344 by Vallée-Bélisle et al. describes the use of a unimolecular probe for detecting and quantifying macromolecules and other analytes. The technology described therein is based on conformational modification of the unimolecular probe upon binding to the target for triggering a detectable signal. However, this technology is limited to the detection of targets or a combination of targets capable of binding simultaneously to two distinct target-binding moieties on the unimolecular probe to induce a conformational modification. The publication of Lass-Napiorkowska et al. (2012) describes a combination of three oligonucleotides which produce a FRET signal upon binding to the target. However, much like the PCT application to Vallée-Bélisles described above, this technology is limited to the detection of targets or a combination of targets capable of binding simultaneously two target-binding moieties present on distinct oligonucleotides to induce the FRET signal. International application WO 2007/120299 by Xiao et al. describes the use of a signal-on architecture for detecting targets. However, this technology is limited to the detection of targets which can specifically bind a nucleic acid molecule. The publication of Fan et al. (2003) describes the use of a single stem-containing oligonucleotide which changes its conformation upon binding to the target. Much like the PCT application to Xiao et al. described above, this technology is limited to the detection of targets which can bind to nucleic acid molecules.
It would be highly desirable to be provided with an assay for the detection/quantification of various targets (which may not necessarily be able to bind to two distinct target-binding moieties or to nucleic acid molecules). It would also be desirable with an assay for the detection/quantification of targets in complex mixtures, such as whole blood and food.